1. ORGANIC STRUCTURE ANALYSIS INTRODUCTION Prof Phil Crews, Dr Jaime Rodriguez and Dr Marcel Jaspars

3. STRUCTURAL DRAWINGS 2D FRAMEWORK 1CARBON SKELETON 2FUNCTIONAL GPS 3LOCATION OF FGs 3D FRAMEWORK 1 RELATIVE STEREO 2 ABSOLUTE STEREO 3 CONFORMATIONAL FEATURES OK But what about:

4. VERIFYING OR CREATING STRUCTURAL DRAWINGS 1. A COMMON TASK 2. MANY ELEMENTS 3. STEREO STRUCTURES 4. CONCISE APPROACHES

5. FOUR TECHNIQUES NMR MS IR UV-VIS DESIRED OUTCOMES MOLECULAR FORMULA FUNCTIONAL GROUPS CH, CC, CZ, OR RINGS FUNCTIONAL GROUP POSITIONS REGIOCHEMISTRY STEREOCHEMISTRY 1)OBTAIN DATA 2)INTERPRET SPECTRA 4. CONCISE APPROACHES

6. REQUIREMENTS FOR A CONCISE APPROACH STEP-BY-STEP ANALYSIS UNDERSTANDING OF BASIC ORGANIC CHEM KNOW COMMON FUNCTIONAL GROUPS

7. REQUIREMENTS FOR A CONCISE APPROACH STEP-BY-STEP ANALYSIS KNOW STABLE VS. UNSTABLE STRUCTURES DEALING WITH ALTERNATIVES

8. 3 TYPES OF PROBLEMS COMMERCIAL SAMPLES - VERIFYING THE LABEL SYNTHETIC REACTION PRODUCT - VERIFYING THE COURSE OF A REACTION UNKNOWN NATURAL PRODUCT - ESTABLISHING A NEW CHEMOTYPE -MOST DIFFICULT TYPE OF APPLICATION -O.S.A. PRINCIPLES & BIOGENETIC, TAXONOMIC ASSUMPTIONS

9. SOME MARINE NATURAL PRODUCTS….

10. WHAT ABOUT PALYTOXIN? MW = 2677 MF = C 129 H 223 N 3 0 54 1 ST ISOLN. 1960’s 1 ST STRUCTURE 1980’s P 2

11. PALYTOXIN MW = 2677 MF = C 129 H 223 N 3 0 54 UN = 20 1 ST ISOLN. 1960’s 1 ST STRUCTURE 1980’s LD 50 = 0.025  g/kg Rabbit LD 50 = 0.45  g/kg Mouse WHAT STRUCTURAL INFO FROM NMR?

12. A solvable problem

13. RELATIVE MERITS OF TECHNIQUES DATA MS 29 PEAKS IR12 PEAKS 1H NMR1 PEAK 13C NMR1 PEAK UV/VISO PEAKS More is less??

15. USES OF MOLECULAR FORMULA OR PARTIAL FORMULA UNSATURATION NUMBER (UN) OR DOUBLE BOND EQUIVALENTS (DBE) C n H 2n+2 ADD H FOR EACH X ADD CH FOR EACH N OR P EXAMPLES UN = 1 C 6 H 12 O EVEN H COUNT C 6 H 11 Cl ODD H COUNT C 6 H 13 N ODD H COUNT C 6 H 14 N 2 EVEN H COUNT

16. USES OF MOLECULAR FORMULA OR PARTIAL FORMULA UNSATURATION NUMBER & FUNCTIONAL GROUP CATEGORIES SELECTED EXAMPLES UN = 0 C-C NOT A FUNCTIONAL GROUP UN = 1 RING, ALKENE, CARBONYL, IMINE, NITRO UN = 2TWO RINGS, POLYENE, CUMULENE, ALKYNE NITRILE, ISONITRILE, ANHYDRIDE UN = 3NON-BENZENOID AROMATICS UN = 4ARENE, PYRIDINE

17. UN = [(2a+2) – (b-d+e)] 2 C 2 H 3 O 2 Cl = (4+2)-3-1 2 = 1 CaHbOcNdXeCaHbOcNdXe Unsaturation number/Double bond equivalents

18. This is the sum of the number of multiple bonds of all kinds kinds (C=C, C  C, C=O, C=N, C  N, N=O, etc) and rings in a molecule, and is extremely useful in structure determination. A double bond has a dbe = 1, a triple bond has a dbe = 2, and a benzene ring has a dbe = 4 (three double bonds plus one ring) Halogens count as hydrogen for this purpose. The number of oxygen atoms is immaterial. If you can identify securely by spectroscopic or other means the number of multiply bonded functional groups, e.g. carbonyl, present, the remainder will be rings. It is therefore possible to decide if the compound is acyclic, monocyclic, bicyclic, etc. and this is extremely helpful in imagining a structure. Once you have obtained the molecular formula, the first step in a structure determination is to work out the dbe.

19. NOW THE QUESTION HOW MANY STRUCTURES FIT C 2 H 3 O 2 Cl ?

21. Limitations in Organic Structure Analysis Horror Stories “Even seasoned investigators sometimes experience difficulties in analyzing the structures of organic compounds”…..

22. BUT

23. Limitations in Organic Structure Analysis More Horror Stories

24. SUBSTRUCTURES – A same Z E not same & D, C, B order different JMC: A-D-C-B-Z-E JACS: A-B-C-D-Z-E

25. [3.2.0] [3.1.1] Limitations in Organic Structure Analysis More Horror Stories

26. Limitations in Organic Structure Analysis Final Horror Story

27. Chemical shift addivity Predicting carbon chemical shifts

28. 21.9 29.7 31.7 11.4 ALIPHATICS: CARBON SHIFTS GENERAL (SP 3 )  10 – 100 UNFUNCTIONALIZED  10 - 50

29. 21.9 29.7 31.7 11.4 ALIPHATICS: CARBON SHIFTS GENERAL (SP 3 )  10 – 100 UNFUNCTIONALIZED  10 - 50 SHIFTS ARE ADDITIVE

30. ADDITIVE PATTERNS

31. UNDERSTANDING THE TABLE 4 3 2 1     1+20 2+10 3 -2 to -3 4  0 also 31 61 +3 

32. SHOW 13 C PREDICT!

33. FUNCTIONAL GROUP ID

34. PROBLEM SOLVING

37. Coupling/Splitting Two nuclei placed close together in the same molecule will interact and the NMR lines will be split into several components. The splitting is also referred to as coupling and the resultant splitting or coupling patterns are also called multiplets. The distance between the members of each multiplet in Hz is the coupling constant J, and can be calculated from the spectrum: J (Hz) =  (ppm) x spectrometer frequency in MHz The proton proton interaction is transmitted through the intervening (s) electrons making up the chemical bonds, so the magnitude of J is an indication of the number and type of bonds.

38. Coupling/Splitting No coupling Coupling aa bb J ab

39. FACTORS CONTROLLING J’s 1)# OF INTERVENING BONDS (1,2,3,4, etc) 2)e - DENSITY 3)ANGLE H-C-C H-C-C-H H-C-C-C H-C-C-C-H 4)BOND HYBRIDIZATION 5)ELECTRONEGATIVTY OF ATOMS ALONG PATH

40. A FOCUS ON FIRST ORDER

41. COMPLEX 1 ST ORDER

42. A FOCUS ON COMPLEX FIRST ORDER

43. TERMS 1.BIG  A/X 2.1 ST ORDER AMX 3.NON 1 ST ORDER AB 4.SPECIAL A/A’ 5.COUPLING 1 J CH 6.VALUES IN Hz 7.2-5 NUCLEI “21 CASES”

44. NON 1 ST ORDER SPIN SYSTEMS 1.AB #2 2.ABC #3 3.A 2 B #4 4.ABCX #8 5.A 2 B 2 #9 6.AA’XX’ #10

45. P 130

46. Given the 1 H NMR chemical shifts and coupling constants for allyl alcohol, explain the observed spectrum (OH peak omitted)

47. MASS SPECTROMETRY

48. Mass Spectrometry MS is an analytical method in which molecules are ionised, and the ions are separated according to their mass. The technique is used to determine molecular weights and hence molecular formulas. MS involves chemical reactions and is therefore not a true spectroscopic method. However, it does complement information provided by: TechniqueWhat is measuredEM radiation?Amount needed NMRnuclear spin transitionsyes1-10 mg IRbond vibrationsyes<1 mg UVelectronic transitionsyes<1 mg MSmass to charge rationo<1 mg

49. Processes in MS MS involves three distinct processes: –Volatilisation of the sample –Ionisation of the molecules –Separation and detection of the ions according to m/z ratio Sample inlet Source Mass analyser Detector Data System Vacuum System Ionisation Mass separation

50. Ionisation Both positive and negative ions can be formed by MS techniques, but positive ions are most commonly used. The ions usually have one positive charge. There are several methods of ionisation, but the most common these days are electrospray (ESI, small/large molecules) and matrix- assisted laser desorption (MALDI, large molecules)

51. Electron Impact (EI) When molecules in the gas phase are bombarded by a stream of electrons, an electron can be ejected to give the molecular ion (M +. ). This technique is the oldest method of forming cations. The lower energy species (M +. ) is much more favoured. Electrons have energies 10-70 eV (960 - 6720 kJ/mol), so the molecular ions formed are highly activated, leading to fragmentation. M +. → Fragments (at > 10 eV)

52. Electrospray (ESI) This is the most popular technique for ionising samples soluble in polar solvents (H 2 O, MeOH and EtOH). It is therefore very important for the analysis of biomolecules of high molecular weight. A derivative of this technique, atmospheric pressure chemical ionisation (APCI) can be used to ionise samples soluble in organic solvents (CH 2 Cl 2, hexanes etc). The main problems arise from the existence of multiply charged ions (which is an advantage for large molecules!), and the presence of other quasi-molecular ions: [M + MeOH + H] +, [M + Na] +, [M + K] +

53. Electrospray (ESI) The dissolved sample is pumped through a steel capillary at a rate of 1-20 mL/min which is at +3 kV. The electric field at the tip of the capillary charges the surface of the emerging droplets. The solvent is evaporated using N 2, reducing the size of the droplet, until the charges come into close proximity, and a Coulombic explosion occurs giving rise to a quasi- molecular ion of the form [M + H] n+ where n can be >1.

54. Electrospray (ESI)

55. Matrix Assisted Laser Desorption (MALDI) Enables gentle ionisation of very large molecules with a single charge Compound dissolved in matrix and deposited in ‘target’ Irradiated by laser beam – energy absorbed by matrix and transferred to analyte which is desorbed.

56. Mass Analysis There are many types of mass analyser: –Magnetic sector/electric sector –Quadrupole –Ion Trap –Time-of-flight –Fourier Transform Ion Cyclotron Resonance (FT- MS) –Orbitrap

57. Magnetic Sector

58. The ions formed are accelerated by a voltage V, and if they have mass m, velocity v and charge z (z is the number of electronic charges). Kinetic energy = potential energy ½mv 2 = zV When a charged particle travels through a magnetic field B, it will travel in a circle with radius r. The equation relating these parameters is: Combining these two equations gives: This allows us to calculate the mass to charge ratio (m/z) of any ion M +.. Since the charge z is usually 1, it is effectively the molecular mass we are measuring. This is extremely useful when trying to determine the molecular formula of a molecule.

59. Monoisotopic masses For many elements, there is more than one isotope, each with a different natural abundance. For the most common elements in organic molecules: IsotopeMass% Abundance 1H1H1.00899.99 12 C12.00098.89 13 C13.0031.11 14 N14.00399.64 15 N15.0000.36 16 O15.99599.76 18 O17.9990.20 32 S31.97295.03 33 S32.9710.76 34 S33.9684.20 35 Cl34.96975.77 37 Cl36.96624.23 79 Br78.91850.52 81 Br80.91649.48

60. Average atomic masses For instance the atomic mass of 12 C is 12.000 and of 13 C is 13.003, but the average atomic mass must take into account the abundance of each isotope. Average atomic mass = Similarly the average atomic mass of Cl is given as 35.5 These are used for mole calculations MS measures monoisotopic masses and these must be used to calculate molecular masses in MS.

61. Calculating Molecular Formulae Using Low Resolution Mass Spectra Rule of 13: CH = 13 amu. Most common unit in organic compounds: Therefore (CH) 10 H 6 = C 10 H 16 Use CH 4 = H 16 = O CH 2 = H 14 = N H 12 = C Gives: C 9 H 12 O, C 8 H 8 O 2, C 8 H 12 N 2 etc M + (molecular ion) = 136 m/z

62. Accurate Mass Measurement Experimental accurate mass measurement (from MS) was 136.1256 suggesting C 10 H 16 is the correct formula. The error between calculated and experimental mass is: 136.1256 - 136.1248 = 0.0008 = 0.8 mmu FormuladbeAccurate mass C 10 H 16 3136.1248 C 9 H 12 O4136.0885 C8H8O2C8H8O2 5136.0522 C7H4O3C7H4O3 6136.0159 C 9 H 14 N3.5136.1123 C 8 H 12 N 2 4136.0998

63. Determining the Molecular Formula Using NMR and MS Data CH CH 2 CH 3 DEPT-135

64. Isotope Ratio Patterns For hydrocarbons, the height of the [M + 1] peak is given by the expression: –[M + 1] = n x 1.1% –where n is the number of carbon atoms. The height of the [M + 1] peak can therefore be used as a crude measure of the number of carbon atoms in a hydrocarbon. For most other compounds, [M + 2] is very small, apart from those containing Cl or Br. The region around the molecular ion peak becomes more complex if either of these (or both) are present.

65. Cl and Br 2 Br atoms in the same molecule: (1 79 Br + 1 81 Br) 2 = 1 79 Br 79 Br + 2 79 Br 81 Br + 1 81 Br 81 Br 2 Cl atoms present: (3 35 Cl + 1 37 Cl) 2 = 9 35 Cl 35 Cl + 6 35 Cl 37 Cl + 1 37 Cl 37 Cl

66. Multiply Charged Ions

67. Using electrospray ionization it is common to get multiply charged ions: [M + H] + [M + 2H] 2+ [M + 3H] 3+ [M + nH] n+ m/z: M + 1 (M+2)/2(M+3)/3(M+n)/n